Systems Analysis of Cardiac Chromatin Structure

描述(申请人提供)：体内间期基因组是什么样子是未知的。在过去的十年中，测序的进展为序列变异(及其表达)提供了新的见解；然而，除了众所周知的有丝分裂的阴谋之外，基因组是如何在三维中组织的，才刚刚开始被理解。成年心肌细胞主要存在于间期，但能够在基因表达上发生大规模变化。转录因子、组蛋白修饰酶和RNA聚合酶复合体在全球基因表达过程中发挥着核心作用；然而，同样重要但研究较少的因素是内源性染色质结构。要被转录，基因的局部环境必须能够与蛋白质结合。这笔赠款的目的是了解这种现象是如何在全基因组范围内整合的：在基本条件下，基因组如何适当地准备好打开和关闭正确的基因，以及在刺激后(例如，在疾病期间)，全球重组染色质的机制是什么？心力衰竭涉及大规模的基因表达变化，包括在发育过程中通常沉默的基因重新激活。我们来自压力超负荷的活体小鼠模型的数据显示，来自连接器组蛋白H1和高迁移率组(HMG)B家族的染色质结构蛋白的丰度和定位发生了变化，并表明心力衰竭与染色质环境的全球重新编程有关，以实现基因激活。我们试图发现HMG和连接子组蛋白如何控制大量染色质重排和全球基因表达的普遍原理；因此，我们将使用斑马鱼、分离的心肌细胞和小鼠心脏作为模型系统。我们的统一假设是，心力衰竭时染色质结构的整体重组是染色质结构蛋白的丰度、基因组定位和蛋白质相互作用发生系统性变化的结果。我们将研究连接器组蛋白和HMG如何在心脏核中建立基因组的高阶结构，以及它们如何在疾病中动态重新包装染色质。我们将使用获得/功能丧失的方法，结合超分辨率STED显微镜(图像染色质包装)、染色质免疫沉淀和DNA测序(定位整个基因组的蛋白质)和蛋白质组学(确定靶向所需的蛋白质)。我们的短期目标是了解HMG和连接子组蛋白在心脏表型中的作用。长期目标是了解HMG、连接子组蛋白和其他染色质结构蛋白如何协调基因组结构，以促进基因表达的特异性。在基础领域的意义在于开发一种完整的染色质包装模型。对临床领域的意义是为基因组如何在疾病中重新编程提供一个机制基础，以便未来的治疗可以针对特定的染色质重塑事件。

DESCRIPTION (provided by applicant): What the interphase genome looks like in vivo is unknown. Advances in sequencing over the last decade have provided new insight into sequence variants (and their expression); yet how the genome is organized in three dimensions, apart from the well-described machinations of mitosis, is only beginning to be understood. Adult cardiac myocytes reside primarily in interphase but are capable of large-scale changes in gene expression. Transcription factors, histone modifying enzymes and RNA polymerase complexes play a central role in the process of global gene expression; however, an equally important and less explored factor is endogenous chromatin structure. To be transcribed, a gene's local environment must be accessible for protein binding. The objective of this grant is to understand how this phenomenon is integrated on a genome- wide scale: how is the genome appropriately poised to have the right genes on and off under basal conditions, and what are the mechanisms that globally reorganize chromatin following a stimulus (e.g. during disease)? Heart failure involves large-scale gene expression changes, including reactivation of genes normally silenced during development. Our data from an in vivo mouse model of pressure overload show alterations in abundance and localization of chromatin structural proteins from the linker histone H1 and high mobility group (HMG) B families, and indicate that heart failure is associated with global reprogramming of the chromatin environment for gene activation. We seek to discover universal principles for how HMGs and linker histones control bulk chromatin rearrangement and global gene expression; therefore, we will employ zebrafish, isolated myocytes and mouse hearts as model systems. Our unifying hypothesis is that global reorganization of chromatin structure during heart failure is the result of systematic changes in the abundance, genomic localization, and protein interactions among chromatin structural proteins. We will examine how linker histones and HMGs establish the higher order structure of the genome in the cardiac nucleus and how they dynamically repackage chromatin in disease. We will use gain/loss-of-function approaches combined with super resolution STED microscopy (image chromatin packing), chromatin immunoprecipitation and DNA sequencing (localize proteins across the genome) and proteomics (determine proteins necessary for targeting). Our short-term goal is to understand the role of HMGs and linker histones in cardiac phenotype. The long-term goal is to develop an understanding of how HMGs, linker histones and other chromatin structural proteins coordinate genomic structure to facilitate specificity in gene expression. The significance in the basic realm is to develop an integrated model of chromatin packing. The significance to the clinical realm is to provide a mechanistic basis for how the genome is reprogrammed with disease, such that future therapies can target specific chromatin remodeling events.